Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The genome of brome mosaic virus (BMV) is comprised of three (+) strand RNAs, each containing a similar, highly structured, 200 base long sequence at its 3' end. A 134 base subset of this sequence contains signals directing interaction of the viral RNA with BMV RNA replicase, ATP,CTP:tRNA nucleotidyl transferase and aminoacyl tRNA synthetase. A series of mutants containing deletions within this region, previously constructed and tested in vitro for the effect on replication and aminoacylation activities, has now been assayed in vitro for adenylation function and in vivo for ability to replicate in isolated protoplasts and whole plants. These tests indicate that features of viral RNA recognized by BMV replicase overlap those directing adenylation, but are distinct from those directing aminoacylation. Consequently, the lethality of a deletion preferentially inhibiting aminoacylation suggests that this function may have an essential role contributing to viral replication in vivo. An RNA3 mutant bearing a 20-base deletion yielding normal levels of aminoacylation and enhanced levels of replicase template activity and adenylation in vitro was able to replicate in protoplasts and plants; however, its accumulation in protoplasts was reduced relative to wild-type. This suggests that additional functions affecting the replication and accumulation of viral RNA reside in the conserved 3' sequence.
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PMID:Modulation of replication, aminoacylation and adenylation in vitro and infectivity in vivo of BMV RNAs containing deletions within the multifunctional 3' end. 375 26

Previous data demonstrated that reovirus mRNA, synthesized in vitro with the particulate RNA transcriptase of reovirus cores, efficiently directs the synthesis of polypeptides in vitro. The present studies indicate that all of the three size classes of reovirus mRNA produced in vitro can form protein initiation complexes with rat liver [(36)S]Met-tRNA(F) and incubated 40S and 60S ribosomal subunits, which had been washed in 0.5 M KCl of mouse fibroblast L-929 cells. Mild prior treatment of the mRNA with HCHO was required to expose the initiator region. The initiation complex reacted quantitatively with puromycin to form a puromycin peptide, whose electrophoretic properties were identical to methionyl-puromycin formed in response to poly(A,G,U) or the initiator codon AUG. The complex was relatively stable and specific for [(35)S]Met-tRNA(F); rat liver [(35)S]Met-tRNA(M) was unreactive unless the supernatant factors EF T(1) and EF T(2) were also present. However, the addition of fusidic acid, at a concentration that did not affect complex formation with [(35)S]Met-tRNA(F), completely inhibited Met-tRNA(M) utilization. Exogenous ribosomal factors and GTP were not required unless the separated 40S and 60S subunits were further treated with 1 M KCl. The data suggest that reovirus mRNA contains AUG initiator codons that form a complex with Met-tRNA(F) at a puromycin-reactive site on ribosomes.
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PMID:Formation of a mammalian initiation complex with reovirus messenger RNA, methionyl-tRNA F , and ribosomal subunits. 450 35

Reverse transcriptase isolated from avian myeloblastosis virus (AMV) and Rauscher murine leukemia virus (RLV) were examined for their ability to catalyze polymerization, ribonuclease H, pyrophosphate exchange, and pyrophosphorolysis reactions. A detailed characterization and a study of requirements for the expression of pyrophosphate exchange and pyrophosphorolysis reactions indicated that a variety of RNA and DNA template-primers supported these catalytic reactions. Furthermore, hydrogen bonding of template to primer was essential, although RNA:RNA template-primers, e.g. poly(rA) . (rU)9 or 70 S RNA . tRNA complex, were not utilized for these reactions. AMV enzyme required Mg2+, and RLV enzyme Mn2+, as the preferred divalent metal ion for the expression of these activities. Response of various catalytic reactions to site-specific inhibitors revealed that polymerization and pyrophosphate exchange reactions were susceptible to reagents that affected either the substrate or the template binding site, intrinsic zinc, or sulfhydryl groups. RNase H and pyrophosphorolysis activities, on the other hand, exhibited susceptibility only to the template site-specific reagent. We, therefore, conclude that RNase H and pyrophosphorolysis reactions are catalyzed through the template binding site while polymerization and pyrophosphate exchange reactions require additional participation of the substrate binding site, as well as that of intrinsic zinc and the presence of reactive sulfhydryl groups.
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PMID:Enzymatic activities associated with avian and murine retroviral DNA polymerases. Catalysis of and active site involvement in pyrophosphate exchange and pyrophosphorolysis reactions. 615 89

Reverse transcriptase from avian myeloblastosis virus can react with periodate-treated primer tRNATrp (beef) to form a Schiff's base between an epsilon-NH2 lysine group within the active center of the enzyme and the dialdehyde derivative of the 3' terminal ribose of tRNA. In the presence of cyanoborohydride the reversible imminium moiety of the Schiff's base is reduced to a more stable adduct. Non-primer tRNAs were not able to reduce the extent of primer fixation to the enzyme. Complete inactivation of the enzyme was attained when the ratio enzyme:tRNA in the complex was 1:1. When the 1:1 adduct was analyzed by polyacrylamide gel electrophoresis, radioactivity from the terminal adenosine of tRNA was found exclusively associated with the alpha subunit. At longer times of labeling the beta subunit was also found linked to the oxidized primer tRNA.
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PMID:Study of the interactions between avian myeloblastosis virus reverse transcriptase and primer tRNA. Affinity labeling and inactivation of the enzyme by periodate-treated tRNATrp. 616 Apr 74

A remarkable feature of the genomic RNAs of several plant viruses is the presence at the 3' end of a region that exhibits tRNA-like functions, including aminoacylation. The three genomic and single subgenomic RNAs of brome mosaic virus (BMV) accept tyrosine in vitro and in vivo, the smallest 3' fragment that can be aminoacylated being about 135 nucleotides long. The roles of the tRNA-like properties are incompletely understood, but an involvement in replication rather than translational functions is likely. We have recently shown (J.J.B. et al., in preparation) that the features recognized by the BMV RNA-specific RNA-dependent RNA polymerase (replicase) for the use of BMV RNA for complementary strand synthesis also lie within the tRNA-like structure. To distinguish between the roles of BMV RNA as a substrate for tyrosyl-tRNA synthetase and BMV replicase, we have now produced BMV RNAs with mutations at two separate loci within the tRNA-like structure. This has been achieved by transcription in vitro from specifically mutagenized cDNA, an approach permitting the generation of targeted mutants without regard to their viability in vivo.
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PMID:Mutant viral RNAs synthesized in vitro show altered aminoacylation and replicase template activities. 647 77

One of the four subunits of bacteriophage Q beta RNA replicase is elongation factor Tu (EF-Tu), the host aminoacyl-tRNA (AA-tRNA) binding protein. To determine whether the RNA polymerase activity requires the tRNA binding site of EF-Tu, we reconstituted replicase with EF-Tu . GTP covalently bound to AA-tRNA. This cross-linked ternary complex (XLTC) was formed by the reaction of N epsilon-bromoacetyl-Lys-tRNA with EF-Tu-GTP. In an EF-Tu-dependent system for the reconstitution of replicase, XLTC restored polymerase activity at least as well as an equivalent amount of EF-Tu. Replicase reconstituted with XLTC was resolved from replicase containing EF-Tu by chromatography on phosphocellulose, a result which confirmed that the tRNA moiety was incorporated into the enzyme. Chromatographic analysis of reconstitution mixtures revealed that XLTC was incorporated into replicase as extensively as EF-Tu. From these results, it appears that the AA-tRNA binding site on EF-Tu is not required for the assembly or activity of Q beta RNA replicase. Furthermore, because the tRNA macromolecule is cross-linked to His-66 of the EF-Tu, the region surrounding His-66 must normally be exposed on the surface of the replicase.
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PMID:Transfer RNA cross-linked to the elongation factor Tu subunit of Q beta replicase does not inhibit Q beta RNA replication. 701 63

The effect of polyamines on Q beta and MS2 phage RNA-directed synthesis of three kinds of protein in an Escherichia coli cell-free system has been studied. With both phage RNAs, the degree of stimulation of protein synthesis by spermidine was in the order RNA replicase greater than A protein, while the synthesis of coat protein was not stimulated significantly by spermidine. The synthesis of RNA replicase was stimulated by 1 mM spermidine approx. 8-fold. From the results of Q beta RNA direct alanyl-tRNA and seryl-tRNA binding to ribosomes and initiation dipeptide synthesis, it is suggested that the preferential stimulation of the synthesis of RNA replicase by spermidine is due at least partially to the stimulation of the initiation of RNA replicase synthesis.
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PMID:Differential stimulation by polyamines of phage RNA-directed synthesis of proteins. 703 95

We show that the reverse transcriptase (RT) encoded by the Mauriceville mitochondrial plasmid of Neurospora closely resembles viral RNA-dependent RNA polymerases in initiating cDNA synthesis opposite the penultimate C residue of a 3' tRNA-like structure and has the unprecedented ability for a DNA polymerase to initiate DNA synthesis at a specific site in a natural template without a primer. The Mauriceville plasmid enzyme can also use DNA or RNA primers in a manner suggesting how a primitive RT could have evolved from an RNA-dependent RNA polymerase into retroviral and other types of RTs. The characteristics of the Mauriceville plasmid RT suggest that it may be related to the progenitor of present-day RTs and DNA polymerases.
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PMID:The Mauriceville plasmid reverse transcriptase can initiate cDNA synthesis de novo and may be related to reverse transcriptase and DNA polymerase progenitor. 750 2

The gypsy group of long-terminal-repeat retrotransposons contains elements having the same order of enzyme domains in the pol gene as do retroviruses. Elements in the gypsy group are now known from yeast, filamentous fungi, plants, insects, and echinoids. Reverse transcriptase and RNase H amino acid sequences from elements in the gypsy group--including the recently described SURL elements, TED, Cft1, and Ulysses,--were aligned and analyzed by using parsimony and bootstrapping methods, with plant caulimoviruses and/or retroviruses as outgroups. Clades supported at the 95% level after bootstrapping include (1) 17.6 with 297 and (2) all of the SURL elements together. Other likely relationships supported at lower bootstrap confidence intervals include (1) SURL elements with mag, (2) 17.6 and 297 with TED, and this collective group with 412 and gypsy, (3) Tf1 with Cft1, (4) IFG7 with Del, and (5) all of the retrotransposons in the gypsy group together, to the exclusion of Ty3. In contrast with an earlier analysis, our results place mag within the gypsy group rather than outside of a cluster that contains gypsy group retrotransposons and plant caulimoviruses. Several features of retrotransposon genomes provide further support for some of the aforementioned relationships. The union of SURL elements with mag is supported by the presence of two RNA binding sites in the nucleocapsid protein. Location of the tRNA primer binding site and the presence of a long open reading frame 3' to the pol gene support the 17.6-297-TED-412-gypsy cluster.
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PMID:Phylogenetic relationships of reverse transcriptase and RNase H sequences and aspects of genome structure in the gypsy group of retrotransposons. 750 45

The fundamental role played by reverse transcriptase in the replication of retroviruses has stimulated the study of the mechanism of action of this enzyme. The reverse transcriptase of the type 1 human immunodeficiency virus forms a stable complex with its cognate transfer RNA replication primer (tRNA(Lys3)). Here, we outline the role of this enzyme in the selection of its primer tRNA, the annealing of primer tRNA to the complementary region of the retroviral genome, and the first attempts to use the reverse-transcriptase-tRNA complex as a new target for antiviral agents.
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PMID:Priming of HIV replication by tRNA(Lys3): role of reverse transcriptase. 751 21


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